Upon infection, T-cell activation and differentiation are initiated through TCR engagement of peptide-MHC molecules on the surface of APCs in the context of co-stimulation and inflammatory cytokines. These cues trigger numerous signal transduction cascades, whose integration is ‘translated’ into changes in gene transcription, protein activity and expression. This ultimately leads to the development of effector function and T-cell-mediated immunity. The MAPK SAPK/JNK cascade plays a major role in regulating a variety of fate decisions including activation, proliferation, differentiation and death. Three genes encode the JNK family members. JNK1 and JNK2 are ubiquitously expressed, whereas the expression of JNK3 is restricted to the brain, heart and testis. Whilst each JNK isoform is ascribed a unique function, how activation of each is independently regulated is not well understood.
Activation of JNK is important for shaping both the innate and adaptive immune response. For innate immune responses, the inflammatory cytokines TNF and IL-1 induce JNK activity. JNK2 and IKKβ induce the production of pro-inflammatory cytokine response to viral dsRNA. Inflammation dependent activation of PLCγ, JNK and NF-κB enhances the ability of dendritic cells and epithelium tissue to induce Th17 responses. JNK signaling is implicated in regulating pro-inflammatory cytokine production, joint inflammation and destruction in rheumatoid arthritis. JNK is also required for polarization of pro-inflammatory macrophages, obesity-induced insulin resistance and inflammation in adipose tissue.
For T lymphocytes, JNK activation plays different roles depending on the T-cell type, the maturation state and the milieu of the responding cell. For example, in developing thymocytes JNK activation appears to have a role in negative selection and the induction of apoptosis, while in mature T cells it regulates the development of effector functions. In mature CD4+ T cells JNKs inhibit Th2 differentiation by suppressing NFAT/JunB signaling and drive Th1 by inducing IL-12Rβ2 expression. Regulation of Treg function through the glucocorticoid-induced tumor necrosis receptor (GITR) also depends on JNK signaling. In addition, JNK1 and JNK2 have distinct functions even within the same type of T cell. For CD8+ T cells, JNK1 functions downstream of the TCR to induce CD25, enabling a proliferative response to IL-2 (FIG. 1). JNK1−/− CD8+ T cells demonstrate enhanced apoptosis in an in vivo anti-viral immune response. By contrast, cells lacking JNK2 are hyper-proliferative due to increased production of IL-2. Furthermore, JNK1 and JNK2 have divergent effects on effector function. JNK1 promotes IFN-γ and Perforin production and optimal killing of tumor cells. Conversely, JNK2−/− CD8+ T cells express more IFN-γ and Granzyme B and exhibit enhanced tumor clearance. Together, these findings illustrate the extreme importance of JNK in an immune response and demonstrate the need to understand the specific regulation of JNK1 and JNK2 to control the outcome of these responses.
The mechanisms that regulate the independent activation of the individual JNK isoforms are poorly understood. The functional specificity of a number of MAPK signaling pathways has been attributed to their regulation by scaffold molecules. Scaffolds provide means for both spatial regulation and network formation that increase the number of outcomes possible when activating a given pathway. Numerous scaffold proteins have been identified for the JNK signaling pathway including β-arrestin-2, CrkII, JIP-1, plenty of SH3s (POSH), and Carma1/Bcl10. Interestingly, Carma1/Bcl10 selectively regulates JNK2 activation in CD8+ T cells. However, the scaffold proteins specific for TCR-mediated JNK1 activation is less clear.
The TCR connects to JNK activation through the guanine exchange factor (GEF) Vav1 and the adaptor/GEF complex, Grb2/SOS. These molecules are recruited to phosphorylated tyrosine residues on the linker for activation of T cells (LAT). Importantly, both Vav1 and Grb2/SOS activate Rac1 and deficiencies in either lead to significant reduction in JNK signaling. POSH (Plenty of SH3) was initially identified as a scaffold protein that linked active Rac1 to JNK and NF-κB activation, while JIP-1 is a scaffold that facilitates JNK activation through the recruitment of MLK and MKK7. Interestingly, in neurons the association of POSH and JIP-1 mediates JNK activation and apoptosis. However, the role of POSH and JIP-1 in TCR-dependent JNK activation is not known.
POSH is a ubiquitously expressed scaffold molecule that assembles components of signaling pathways that lead to regulation of a number of essential cellular functions. Many of the functions are specific to the type of tissue or the maturation state of the cell. Inhibitors are designed to interfere with the assembly of signaling module and block signals.
Here we investigated the role of POSH in JNK activation in CD8+ T cells. Using a peptide inhibitor strategy, we determined that the interaction between POSH and JIP-1 is required for JNK1, but not JNK2, phosphorylation and T-cell effector function. Most interestingly, the disruption of the POSH/JIP-1 complex results in functional defects that pheno-copy JNK1−/− T cells. Un-coupling POSH and JIP-1 resulted in decreased proliferation, defects in IFN-γ and TNF-α expression and markedly reduced tumor clearance. Correspondingly, the POSH/JIP-1 regulation of JNK1 was also important for the induction of the transcription factors c-Jun, T-bet and Eomesodermin (Eomes), which play important roles in programming effector function. Collectively, these data indicate for the first time, that POSH and the POSH/JIP-1 scaffold network is specifically required for JNK1 dependent T-cell differentiation and effector function in mature CD8+ T cells.